Isolation And Identification On BVDV And Cloning, Analysis Of Genetic Variation On NS2-3

Some dejection and blood samples from aborted bovine, which was suspected to be bovine viral diarrhea, were inoculated to MDBK cells, and one virus stain was successfully isolated. It presented itself classical and regular cytopathic effects (CPE) which was the same as those of typical bovine viral diarrhea virus (BVDV) produced. Other infected cells which had no CPE were detected by sandwich ELJSA, and the negative result showed there was no noncytopathic BVDV strain.Exogenous sequence insertion domain of NS2-3 of the isolated virus was amplified by RT-PCR. The obtained fragment was approximate 665bp. There was no insertion in the isolated virus genome.Healthy male calf was inoculated against the MDBK culture of the isolated virus, and was dissected after 25 days. The symptom and pathologic changes were the same as acute bovine viral diarrhea. Spleen, marrow, lymph node and blood of the calf were used to inoculate MDBK cells, and the time of cytopathic effects advanced and the degree aggravated. The infected MDBK culture was detected by RT-PCR, and the expected 665bp fragment was obtained.The product of PCR was cloned into pMD18-T vector, and then transfected into JM109. The recombinant plasmids were extracted and identificated by enzyme digest and PCR. The nucleotide sequence of the isolated virus NS2-3 was sequenced and amino acid sequence was inferred and compared. The result showed the homology of nucleotide sequence and amino acid sequence with VEDEVAC strain was 100%. The isolated virus strain had no exogenous sequence insertion, gene recombination, gene rearrangement or gene deficiency. However, some nucleotide sequences were replaced. All the above results proved this virus was BVDV and was designated VEDEVAC-Like (HB), which belongs to subtype Ib.